Methods for rolling circle amplification and signal trapping of libraries

ABSTRACT

A method for isolating a polynucleotide that encodes a polypeptide of interest which comprises a signal sequence for secretion or partial secretion, utilizing rolling circle amplification and signal trapping of libraries.

FIELD OF INVENTION

A method for isolating genes encoding secreted polypeptides from rollingcircle amplified gene libraries is described, in which the endogenoussecretion signal sequences are detected using an in vitro transpositionreaction, where the transposon comprises a secretion reporter.

BACKGROUND OF THE INVENTION

Secreted polypeptides are highly interesting for the biotechnologicaland pharmaceutical industries, since they can be produced recombinantlywith a minimum of purification steps necessary. A positive screeningsystem which selects only clones encoding secreted polypeptides is thusvery desirable. Signal trapping is used to identify genes encodingpolypeptides that comprise a signal peptide by applying a translationalfusion to an extracellular reporter encoding gene lacking its ownsignal. Methods and protocols for using signal trapping were disclosedin WO 01/77315 (Novozymes A/S).

Cloning and gene library construction using rolling circleamplification, mainly for sequencing purposes of in particularnon-clonable targets, was disclosed by P. M. Lizardi (U.S. Pat. No.6,287,824; U.S. Pat. No. 6,280,949), and by Dean et al. (2001, RapidAmplification of plasmid and phage DNA using Phi29 DNA polymerase andmultiply-primed rolling circle amplification. Genome Research11:1095-1099). Commercial kits are available comprising Phi29 DNApolymerase, for use in rolling circle amplification sequencing, e.g.TempliPhi™ DNA Sequencing Template Amplification Kit (AmershamBiosciences, USA).

The construction of gene libraries for cloning or screeningtraditionally employs intermediate amplification host cells, e.g.Escherichia coli, because the transformation efficiency is very low withraw ligations, especially into eukaryotic host cells. The use ofintermediate host cells for amplifying and/or maintaining a gene libraryincreases the likelihood of the library not being representative of theorganism it was prepared from. There may well be a bias against geneticmaterial, the presence of which could be inhibitory or even lethal tothe intermediate host, e.g. genes encoding anti-microbial activities maybe lost from the library.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention is how to identifythose clones in a gene library that encode efficiently secreted orsurface-displayed polypeptides, even polypeptides with unknown activity,without having to redone the library into a screening-vector, withouthaving to amplify the library in an intermediate host, and withouthaving to screen the library in traditional labour—and time consumingactivity assays that would detect known activities only. Solving thisproblem allows rapid and efficient industrial exploitation of relevant ssecreted or surface-displayed polypeptides from new organisms.

We describe methods for signal trapping a gene library with asignal-less reporter gene, combined with an in vitro rolling circleamplification procedure allowing a sufficiently high transformationefficiency to circumvent the need for intermediate amplification hostcells, thereby enabling the efficient identification and isolation of apolynucleotide encoding a polypeptide of interest which comprises asignal sequence.

Accordingly, in a first aspect the invention relates to a method forisolating a polynucleotide that encodes a polypeptide of interest whichcomprises a signal sequence for secretion or partial secretion, themethod comprising the sequential steps of:

-   -   a) providing a DNA or cDNA library from an organism producing        the polypeptide of interest, wherein the library is comprised in        a circular vector and is produced in vitro without ultraviolet        irradiation of the component polynucleotides;    -   b) amplifying the library by rolling circle amplification,        thereby forming concatamers;    -   c) inserting into the library a DNA fragment comprising a        promoterless and secretion signal-less polynucleotide encoding a        secretion reporter;    -   d) introducing the amplified library comprising the inserted DNA        fragment into a host cell;    -   e) screening for and selecting a host cell that secretes or        partially secretes the active secretion reporter; and    -   f) identifying from the selected host cell the polynucleotide        into which the secretion reporter was inserted, and isolating        the polynucleotide;        wherein steps b) and c) may be performed in any order.

The terms “secretes”, “partially secretes”, or “membrane displayed” areused interchangeably herein and mean translocation of a part of apolypeptide or of a whole polypeptide across a membrane of a cell suchas a prokaryotic, eukaryotic, or archaea cell. In a non-limiting exampleof polypeptide secretion, a membrane-bound or transmembrane protein suchas a receptor may in the method of the invention be expressed in a hostcell as a fusion polypeptide that is fused with the “secretion reporter”of the invention; thus “secretion” in this context means translocationof the fusion polypeptide across a membrane of the host cell to such anextent that at least the secretion reporter part of the fusionpolypeptide is displayed on the extracellular side of the membrane andis functionally active in a secretion reporter assay. In other examplesthe fusion polypeptide may be completely secreted into the cultivationmedia without any residual linkage to the secreting cell.

In a non-limiting example herein, cDNA or genomic DNA libraries aretagged with a transposon containing a reporter gene. All in-framefusions of the transposon reporter gene with a gene in the librarycontaining a signal sequence are detected by assaying the expression ofactive reporter. The upstream and downstream flanking DNA sequences ofthe transposon insertion are then sequenced and the gene into which thetransposon was inserted is identified by sequence analysis. In manycases, obtaining the full sequence of a tagged gene will be facilitatedby the recovery of numerous clones of the same gene tagged in differentnucleotide positions or sites. Positive clones are sequenced to identifyclones that represent the same gene but have different transposoninsertion sites. In this way all or most of the open reading frame (ORF)can be obtained by contig assembly. If a complete ORF cannot be obtainedin this manner, perhaps due to an insufficient number or an unevendistribution of transposon inserts in the gene, then the full lengthgene may be obtained by classical primer walking DNA sequencing.

The sequence information thus obtained can then be used to isolate thecomplete gene of interest including the sequence encoding the secretionsignal sequence and further to make an optimal expression construct forindustrial production of the secreted proteins, all well within theskill of the art, whereafter the industrial production process ofexpressing and recovering the enzyme is a matter thoroughly described inthe art as shown elsewhere herein.

In a second aspect the invention relates to a polynucleotide encoding apolypeptide of interest, wherein said polynucleotide is isolated by themethod of the present invention. A third aspect of the invention relatesto a polypeptide of interest which is encoded by a polynucleotide asdefined in the second aspect. A fourth aspect relates to an expressionsystem comprising a polynucleotide as defined in the second aspect. In afifth aspect the invention relates to a host cell comprising at leastone copy of a polynucleotide as defined in the second aspect, or anexpression system as defined in the fourth aspect. A final aspectrelates to a process for producing a polypeptide of interest, comprisingcultivating a host cell as defined in the previous aspect underconditions suitable for expressing the polynucleotide as defined in thesecond aspect, wherein said host cell secretes the polypeptide encodedby said polynucleotide into the growth medium.

DEFINITIONS

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989“) DNA Cloning: A Practical Approach, Volumes Iand II/D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed.1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds(1985)); Transcription And Translation (B. D. Hames & S. J. Higgins,eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, APractical Guide To Molecular Cloning (1984).

When applied to a protein, the term “isolated” indicates that theprotein is found in a condition other than its native environment, suchas apart from blood and animal tissue. In a preferred form, the isolatedprotein is substantially free of other proteins, particularly otherproteins of animal origin. It is preferred to provide the proteins in ahighly purified form, i.e., greater than 95% pure, more preferablygreater than 99% pure. When applied to a polynucleofide molecule, theterm “isolated” indicates that the molecule is removed from its naturalgenetic milieu, and is thus free of other extraneous or unwanted codingsequences, and is in a form suitable for use within geneticallyengineered protein production systems. Such isolated molecules are thosethat are separated from their natural environment and include cDNA andgenomic clones. Isolated DNA molecules of the present invention are freeof other genes with which they are ordinarily associated, and mayinclude naturally occurring 5′ and 3′ untranslated regions such aspromoters and terminators. The identification of associated regions willbe evident to one of ordinary skill in the art (see for example, Dynanand Tijan, Nature 316: 774-78, 1985).

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases, the sequence of apolynucleotide is read from the 5′ to the 3′ end. Polynucleotidesinclude RNA and DNA, and may be isolated from natural sources,synthesized in vitro, or prepared from a combination of natural andsynthetic molecules. A “nucleic acid molecule” refers to the phosphateester polymeric form of ribonucleosides (adenosine, guanosine, uridineor cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine,deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”) ineither single stranded form, or a double-stranded helix. Double strandedDNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acidmolecule, and in particular DNA or RNA molecule, refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary or quaternary forms. Thus, this term includesdouble-stranded DNA found, inter alia, in linear or circular DNAmolecules (e.g., restriction fragments), plasmids, and chromosomes. Indiscussing the structure of particular double-stranded DNA molecules,sequences may be described herein according to the normal convention ofgiving only the sequence in the 5′ to 3′ direction along thenon-transcribed strand of DNA (i.e., the strand having a sequencehomologous to the mRNA). A “recombinant DNA molecule” is a DNA moleculethat has undergone a molecular biological manipulation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention allows the screening of genebanks or -libraries byproxy, for genes encoding secreted polypeptides or enzymes even ofunknown activity and thus without known screening assays includingpolypeptides having antimicrobial or biocidal activity. The is methodsof the invention enable screening for polypeptides of potentialindustrial interest that would not likely have been isolated usingconventional screening assays.

A method for isolating a polynucleotide that encodes a polypeptide ofinterest which comprises a signal sequence for secretion or partialsecretion, the method comprising the sequential steps of:

-   -   a) providing a DNA or cDNA library from an organism producing        the polypeptide of interest, wherein the library is comprised in        a circular vector and is produced in vitro without ultraviolet        irradiation of the component polynucleotides;    -   b) amplifying the library by rolling circle amplification,        thereby forming concatamers;    -   c) inserting into the library a DNA fragment comprising a        promoterless and secretion signal-less polynucleotide encoding a        secretion reporter;    -   d) introducing the amplified library comprising the inserted DNA        fragment into a host cell;    -   e) screening for and selecting a host cell that secretes or        partially secretes the active secretion reporter; and    -   f) identifying from the selected host cell the polynucleotide        into which the secretion reporter was inserted, and isolating        the polynucleotide;        wherein steps b) and c) may be performed in any order.

The present invention can be performed using any gene libraries known inthe art, specifically it can also be used with gene libraries of viablebut non-culturable organisms as typically seen in environmental samples.Processes of producing representative or normalized gene-libraries fromenvironmental samples containing non-culturable organisms have beendescribed in the art (U.S. Pat. No. 5,763,239). Accordingly a preferredembodiment of the present invention relates to a method of the firstaspect, wherein the cDNA or the cDNA library is normalized or whereinthe DNA or the cDNA library is normalized.

A preferred embodiment relates to a method of the first aspect, whereingenomic DNA library or cDNA library is derived from a microorganism;preferably the microorganism is a fungus, a filamentous fungus or ayeast; more preferably the microorganism is a bacterium, or themicroorganism is an archaeon.

Another preferred embodiment relates to a method of the first aspect,wherein the genomic DNA library or cDNA library is derived from amulticellular organism, preferably from a mammalian cell most preferablyfrom a human cell.

Vectors

The present invention also relates to recombinant vectors comprising anucleic acid sequence, of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleic acidand control sequences described above may be joined together to producea recombinant expression vector which may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, thenucleic acid sequence of the present invention may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid,phagemid, cosmid, or virus) which can be conveniently subjected torecombinant DNA procedures and can bring about the expression of thenucleic acid sequence. The choice of the vector will typically depend onthe compatibility of the vector with the host cell into which the vectoris to be introduced. The vectors may be linear or closed circularplasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which i has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol or tetracycline resistance. Suitable markers for yeasthost cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectablemarkers for use in a filamentous fungal host cell include, but are notlimited to, amdS (acetamidase), argB (ornithine carbamoyltransferase),bar (phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), as well as equivalents thereof. Preferredfor use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome.

For integration into the host cell genome, the vector may rely on thenucleic acid sequence encoding the polypeptide or any other element ofthe vector for stable integration of the vector into the genome byhomologous or nonhomologous recombination. Alternatively, the vector maycontain additional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 1,500 base pairs, preferably 400 to 1,500base pairs, and most preferably 800 to 1,500 base pairs, which arehighly homologous with the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1permitting replication in Bacillus. Examples of origins of replicationfor use in a yeast host cell are the 2 micron origin of replication,ARS1, ARS4, the combination of ARS1 and CEN3, and the combination ofARS4 and CEN6. The origin of replication may be one having a mutationwhich makes its functioning temperature-sensitive in the host cell (see,e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA75: 1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Since the rolling circle amplification of the invention generatesconcatamers as an end-product, it may be advantageous to convert theconcatamers to monomers. This monomerization may be achieved in anymanner which is convenient, e.g. by introducing at least one restrictionenzyme cleavage site in the vector for subsequent restriction andcircularization; or by introducing at least one cos site to enablecleavage during phage packaging; or by introducing at least onerecombination recognition site, such as the Flp recombinase recognitionsite, for subsequent looping out of monomers using the Flp enzyme.Accordingly, in a preferred embodiment the invention relates to a methodof the first aspect, wherein the vector comprises at least onerestriction enzyme cleavage site and/or at least one cos site and/or atleast one recombination recognition site.

Another preferred embodiment of the invention relates to a method of thefirst aspect, wherein the amplified library concatamers are converted tomonomers before performing step d); preferably the vector comprises atleast one restriction enzyme recognition site, and the concatamers areconverted to monomers by restriction enzyme digestion and thencircularized by ligation, or preferably the vector comprises at leastone recombination recognition site, and the concatamers are converted tomonomers by circularization through homologous recombination, mediatedby the recognition sites and a specific recombinase.

It is well known in the art, that supercoiled circular polynucleotideshave a higher transformation efficiency than their relaxed counterparts.Accordingly, in a preferred embodiment the invention relates to a methodof the first aspect, wherein the monomers are circularized and thentreated with a DNA topoisomerase.

Yet another preferred embodiment of the invention relates to a method ofthe first aspect, wherein the vector comprises at least one cos site,and wherein subsequent to steps b) and c) the amplified libraryconcatamers are converted to monomers prior to step d) by cos sitecleavage during phage-packaging.

In some instances, better transformation efficiency is achieved whenconcatamers are used rather than monomers, e.g. for many Bacillus cellsthis is the case. Accordingly, one preferred embodiment of the inventionrelates to a method of the first aspect, wherein the library isintroduced into the host cell as concatamers.

A DNA fragment comprising a promoterless and secretion signal-lesspolynucleotide encoding a secretion reporter is inserted into thelibrary in step c) of the method of the first aspect. This may be doneby an in vitro process, such as described in WO 01/77315.

So, a preferred embodiment of the invention relates to a method of thefirst aspect, wherein step c) is performed in vitro. A further preferredembodiment of the invention relates to a method of the first aspect,wherein the DNA fragment of comprises a transposon, preferably a MuAtransposon. Still another preferred embodiment of the invention relatesto a method of the first aspect, wherein the DNA fragment comprises anorigin of replication which is functional in the host cell, preferablythe origin of replication is functional in Escherichia coli, morepreferably the origin of replication is a derivative of colE1, oriV,P15A, or colDF1 3, and most preferably the origin of replication iscolE1.

Another preferred embodiment of the invention relates to a method of thefirst aspect, s wherein the secretion reporter is a protein which, whensecreted from the host cell, allows said cell to grow in the presence ofa substance which otherwise inhibits growth of said cell; preferably thesecretion reporter is a β-lactamase or an invertase.

In still another preferred embodiment, the invention relates to a methodof the first aspect, wherein the polynucleotide of the DNA-fragment ofsteb (b) encodes a secretion reporter carrying an N-terminal peptidelinker which comprises a specific target site for proteolytic cleavage.

Host Cells

The present invention also relates to recombinant host cells, which areadvantageously used in the method of the first aspects of the inventionas well as in recombinant production of the polypeptides encoded by thegene of interest identified in the method of the invention. A vectorcomprising a nucleic acid sequence or gene of interest of the presentinvention is introduced into a host cell so that the vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell for these purposes will to a large extent depend upon thegene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, erg., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular cells are bacterial cells such as gram positivebacteria including, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus clausii, Bacillus coagulans, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or aStreptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus,or gram negative bacteria such as E. coli and Pseudomonas sp. in apreferred embodiment, the bacterial host cell is a Bacillus lentus,Bacillus licheniformis, Bacillus stearothermophilus, or Bacillussubtilis cell. In another preferred embodiment, the Bacillus cell is analkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporaton (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may be a eukaryote, such as a mammalian, insect, plant, orfungal cell. In a preferred embodiment, the host cell is a fungal cell.“Fungi” as used herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in is Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra).

In a more preferred embodiment, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9,1980).

In an even more preferred embodiment, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell. In a most preferred embodiment, the yeast host cell is aSaccharomyces carisbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasli, Saccharomyces kluyveri,Saccharomyces norbensis or Saccharomyces oviformis cell. In another mostpreferred embodiment, the yeast host cell is a Kluyveromyces lactiscell. In another most preferred embodiment, the yeast host cell is aYarrowia lipolytica cell.

In another more preferred embodiment, the fungal host cell is afilamentous fungal cell. “Filamentous fungi” include all filamentousforms of the subdivision Eumycota and Oomycota (as defined by Hawksworthet al., 1995, supra). The filamentous fungi are characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative. In an even more preferred embodiment, the filamentousfungal host cell is a cell of a species s of, but not limited to,Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora,Neurospora, Penicillium, Thielavia, Tolypocladium, or Trichoderma.

In a most preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In an even mostpreferred embodiment, the filamentous fungal parent cell is a Fusariumvenenatum (Nirenberg sp. nov.) cell. In another most preferredembodiment, the filamentous fungal host cell is a Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023 andYelton et al., 1984, Proceedings of the National Academy of Sciences USA81: 1470-1474.

Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, Journal ofBacteriology 153: 163; and Hinnen et al., 1978, Proceedings of theNational Academy of Sciences USA 75: 1920.

Accordingly, in a preferred embodiment the invention relates to a methodof the first aspect, wherein the host cell is bacterial; preferably thebacterial host cell is an Escherichia, Lactococcus, Streptomyces,Enterococcus or Bacillus cell, preferably of the species Escherichiacoli, Lactococcus lactis, Streptomyces griseus, Streptomyces coelicor,Enterococcus faecalis, Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus stearothermophilus,Bacillus subtilis, or Bacillus thuringiensis.

In another embodiment the invention relates to a method of the firstaspect, wherein the host cell is fungal; preferably the fungal host cellis of the genus Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, Yarrowia, Acremonium, Aspergillus, Aureobasidium,Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia,Tolypocladium, or Trichoderma; more preferably the fungal host cell isof the species Saccharomyces cerevisiae, Aspergillus aculeatus,Aspergillus awamori, Aspergillus nidulans, Aspergillus niger, orAspergillus oryzae.

In yet another preferred embodiment the invention relates to a method ofthe first aspect, wherein the host cell is mammalian.

When a host cell has been selected according to the method of theinvention, it is of interest to identify the polynucleotide into whichthe secretion reporter was inserted. There are many ways in the art thatallow such an identification, e.g. hybridizations, probing, subcloningetc. An efficient way is to perform a polynucleotide sequencing of theDNA flanking the inserted DNA fragment, the sequence of which is alreadyknown. So, a preferred embodiment of the invention relates to a methodof the first aspect, wherein the identifying of the polynucleotide instep f) is done by DNA sequencing using at least one primer directed tothe DNA fragment of step c), or using at least one primer directed tothe vector of step a); preferably where isolating the polynucleotide instep f) is done by utilizing the DNA sequence information obtained.

Once sequence information has been obtained on the polynucleotideencoding the polypeptide of interest, then the complete encoding genemay be isolated, either from the genome of the originating organism, orfrom the previously established library. Accordingly, a preferredembodiment of the invention relates to a method of the first aspect,wherein the polynucleotide in step f) is isolated from the genome of theorganism producing the polypeptide of interest, or from a DNA or cDNAlibrary of the organism.

A great number of secreted or partially secreted polypeptides are ofcommercial or industrial interest. A preferred embodiment of theinvention relates to a method of the first aspect, wherein thepolypeptide of interest is an enzyme that is secreted from the hostcell.

Another preferred embodiment of the invention relates to a method of thefirst aspect, wherein the polypeptide of interest is a membrane-boundreceptor, preferably a two-component signal (TCS) transduction receptor,and more preferably a cytokine receptor. Yet another preferredembodiment of the invention relates to a method of the first aspect,wherein the polypeptide of interest is a secreted cytokine. Stillanother preferred embodiment of the invention relates to a method of thefirst aspect, wherein the polypeptide of interest is a polypeptide whichelicits an immunogenic response in humans. A preferred embodiment of theinvention relates to a method of the first aspect, wherein thepolypeptide of interest has antimicrobial activity, or wherein thepolypeptide of interest is a plant pathogenic polypeptide.

The industrial route to producing the polypeptide of interest inrelevant quantities will often involve utilizing recombinant expressionsystems. Accordingly, a preferred embodiment of the invention relates toa method of the first aspect, wherein an additional step of constructingan expression system is performed, said expression system comprising thepolynucleotide isolated in step f).

A second aspect of the invention relates to a polynucleotide encoding apolypeptide of interest, wherein said polynucleotide is isolated by themethod of the present invention. A third aspect relates to a polypeptideof interest which is encoded by a polynucleotide as defined in thesecond aspect.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleic acid sequence of the present invention operably linked to oneor more control sequences which direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences. Expression will be understood to include any stepinvolved in the production of the polypeptide including, but not limitedto, transcription, post-transcriptional modification, translation,post-translational modification, and secretion. “Expression construct”,“expression vector, or “expression system” are used interchangeablyherein, and are defined as a nucleic acid molecule, either single- ordouble-stranded, which is isolated from a naturally occurring gene orwhich has been modified to contain segments of nucleic acid combined andjuxtaposed in a manner that would not otherwise exist in nature. Theterm nucleic acid construct is synonymous with the term expressioncassette when the nucleic acid construct contains all the controlsequences required for expression of a coding sequence of the presentinvention. The term “coding sequence” is defined herein as a nucleicacid sequence which directly specifies the amino acid sequence of itsprotein product. The boundaries of the coding sequence are generallydetermined by a ribosome binding site (prokaryotes) or by the ATG startcodon (eukaryotes) located just upstream of the open reading frame atthe 5′ end of the mRNA and a transcription terminator sequence locatedjust downstream of the open reading frame at the 3′ end of the mRNA. Acoding sequence can include, but is not limited to, DNA, cDNA, andrecombinant nucleic acid sequences.

An isolated nucleic acid sequence encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the nucleic acid sequenceprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifying nucleicacid sequences utilizing recombinant DNA methods are well known in theart.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for the expression of a polypeptideof the present invention. Each control sequence may be native or foreignto the nucleic acid sequence encoding the polypeptide. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide. The term “operably linked” is defined herein asa configuration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the DNA sequence such thatthe control sequence directs the expression of a polypeptide.

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcriptionalcontrol sequences which mediate the expression of the polypeptide. Thepromoter may be any nucleic acid sequence which shows transcriptionalactivity in the host cell of choice including mutant, truncated, andhybrid promoters, and may be obtained from genes encoding extracellularor intracellular polypeptides either homologous or heterologous to thehost cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al, 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et at, 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook, J. et al., 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (WO 96/00787), as well as theNA2-tpi promoter (a hybrid of the promoters from the genes forAspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleic acid sequence encoding the polypeptide. Any leader,sequence that is functional in the host cell of choice may be used inthe present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

The present invention also relates to nucleic acid constructs foraltering the expression of an endogenous gene encoding a polypeptide ofthe present invention. The constructs may contain the minimal number ofcomponents necessary for altering expression of the endogenous gene. Inone embodiment, the nucleic acid constructs preferably contain (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, and (d) asplice-donor site. Upon introduction of the nucleic acid construct intoa cell, the construct inserts by homologous recombination into thecellular genome at the endogenous gene site. The targeting sequencedirects the integration of elements (a)-(d) into the endogenous genesuch that elements (b)-(d) are operably linked to the endogenous gene.In another embodiment, the nucleic acid constructs contain (a) atargeting sequence, (b) a regulatory sequence, (c) an exon, (d) asplice-donor site, (e) an intron, and (f) a splice-acceptor site,wherein the targeting sequence directs the integration of elements(a)-(f) such that elements (b)-(f) are operably linked to the endogenousgene. However, the constructs may contain additional components such asa selectable marker.

The introduction of these components results in production of a newtranscription unit in which expression of the endogenous gene isaltered. In essence, the new transcription unit is a fusion product ofthe sequences introduced by the targeting constructs and the endogenousgene. In one embodiment in which the endogenous gene is altered, thegene is activated. In this embodiment, homologous recombination is usedto replace, disrupt, or disable the regulatory region normallyassociated with the endogenous gene of a parent cell through theinsertion of a regulatory sequence which causes the gene to be expressedat higher levels than evident in the corresponding parent cell.

The constructs further contain one or more exons of the endogenous gene.An exon is defined as a DNA sequence which is copied into RNA and ispresent in a mature mRNA molecule such that the exon sequence isin-frame with the coding region of the endogenous gene. The exons can,optionally, contain DNA which encodes one or more amino acids and/orpartially encodes an amino acid. Alternatively, the exon contains DNAwhich corresponds to a 5′ non-encoding region. Where the exogenous exonor exons encode one or more amino acids and/or a portion of an aminoacid, the nucleic acid construct is designed such that, upontranscription and splicing, the reading frame is in-frame with thecoding region of the endogenous gene so that the appropriate readingframe of the portion of the mRNA derived from the second exon isunchanged.

The splice-donor site of the constructs directs the splicing of one exonto another exon. Typically, the first exon lies 5′ of the second exon,and the splice-donor site overlapping and flanking the first exon on its3′ side recognizes a splice-acceptor site flanking the second exon onthe 5′ side of the second exon, A splice-acceptor site, like asplice-donor site, is a sequence which directs the splicing of one exonto another exon. Acting in conjunction with a splice-donor site, thesplicing apparatus uses a splice-acceptor site to effect the removal ofan intron.

A fourth aspect relates to an expression system comprising apolynucleotide as defined in the second aspect. A fifth aspect relatesto a host cell comprising at least one copy of a polynucleotide asdefined in the second aspect, or an expression system as defined infourth aspect.

Many ways have been described in the art to construct host cellscomprising several stable copies of a polynucleotide of interest, bothas independently replicating extrachromosomal entities, and aschromosomally stably integrated copies. A preferred embodiment of thefifth aspect relates to the host cell, wherein at least two copies ofthe polynucleotide as defined in the second aspect are chromosomallyintegrated.

Process of Production

The present invention also relates to processes for producing apolypeptide of the present invention comprising (a) cultivating astrain, which in its wild-type form is capable of producing thepolypeptide, to produce a supernatant comprising the polypeptide; and(b) recovering the polypeptide.

The present invention further relates to methods for producing apolypeptide of the present invention comprising (a) cultivating ahomologously recombinant cell, having incorporated therein a newtranscription unit comprising a regulatory sequence, an exon, and/or asplice donor site operably linked to a second exon of an endogenousnucleic acid sequence encoding the polypeptide, under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The methods are based on the use of gene activationtechnology, for example, as described in U.S. Pat. No. 5,641,670.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermentors performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedure's(e.g., preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see,. e.g.,Protein Purification, J. C. Janson and Lars Ryden; editors, VCHPublishers, New York, 1989).

A final aspect relates to a process for producing a polypeptide ofinterest, comprising cultivating a host cell as defined in the previousaspects under conditions suitable for expressing the polynucleotide ofthe third aspect, wherein said host cell secretes the polypeptideencoded by said polynucleotide into the growth medium. A preferredembodiment relates to the process of the previous aspect, wherein thepolypeptide is an enzyme or a polypeptide having anti-microbialactivity. Another preferred embodiment relates to the process of theprevious aspect, where an additional step of purifying the polypeptideis performed.

EXAMPLES

The vector used herein is denoted pMhas5, and the nucleotide sequence isshown in SEQ ID NO:1. The vector has the following features: FeatureLocation Description CDS  365-1156 Kanamycin resistance CDS 2232-2387Beta galactosidase alpha peptide −10 signal 2189-2192 Shine DalgamoPromoter 2101-2189 Lac promoter misc feature 626-650 KanP1 primer forBACE systemUV Light

Surprisingly, rolling circle amplification worked on plasmid DNAisolated from E. coli but not on the ligation mixture that was used totransform the E. coli. We determined in a series of experiments, thatthe reason for why the ligated DNA was not susceptible to rolling circleamplification, was that pyrimidine dimers were present in the ligatedDNA. The pyrimidine dimers were formed by exposure of the fragment andplasmid DNA with ultraviolet light before ligation.

A plasmid library was diluted to 1 ng/μl DNA in TE buffer (pH8.0). 10 μlof the diluted plasmid pool was spotted onto a UV source (Eagle Eye™ IIUV transilluminator, Stratagene, USA), which was adjusted to preparativemode (UV 360 nm). 2 μl samples were taken every 30 seconds from theirradiated DNA pool, starting with time=0 immediately before turning theUV light on. 10 μl Amersham TempliPhi™ denaturing buffer was then addedto each sample and these were then heated to 95° C. for 3 minutes. 10 μlof TempliPhi™ enzyme mix was then added to each sample and the reactionwas incubated at 30° C. for 8 hours. The samples were subsequentlydenatured at 95° C. for 5 minutes, and then stored at 4° C.

Agarose gel loading buffer was added to the samples which were thenanalyzed in 1% agarose gel electrophoresis according to standardmethods. The results showed that the amount of amplification product wassignificantly reduced in the samples taken after only 30 seconds UVtreatment, decreasing until no amplification product was detectable inthe sample taken after two minutes of UV irradiation. Presumablymodifications of the template DNA had taken place, either by nicking,depurination or pyrimidine dimer formation. The result demonstrated thatit is essential to avoid UV treatment of templates to be used in rollingcircle amplification.

Library Reparation

5 μg of plasmid pMhas5 was restricted with EcoRI and NotI according tothe manufacturers instructions (New England Biolabs, USA). Therestricted plasmid was loaded in a standard 1% TBE agarose gel andelectrophoresed in order to separate the stuffer fragment from thevector. Half of the gel, containing the DNA marker (1 Kb ladder, BRL,USA), and the region corresponding to approximately 1 mm of the 10 mmtotal width of the sample lane, was separated using a ruler as a guide.The separated part of the gel was stained with Ethidium bromide andvisualized on a UV light source. The band corresponding to the cutvector (ca. 2.6 kb) was marked by making a notch in the gel with ascalpel blade. The two gel parts were aligned and the non UV treatedportion corresponding to the cut plasmid was isolated.

A standard GFX™ purification (AP Biotech) was used to remove agarose,restriction enzyme and contaminants. The restricted plasmid was elutedin 50 uls 10 mM Tris buffer pH 8.0 and stored at −20° C. until furtheruse.

A cDNA library was prepared from Rhizomucor pusillus induced for 5 dayson Mex-1 media, as shown in the examples of WO 98/38288 and in theexamples of international patent application WO 01/12794. The protocolsof these PCT publication were used as described, except that care wastaken to eliminate all steps involving UV treatment of the RNA, and DNAduring the standard procedures. No size fractionation was performed onthe RNA, first strand or second strand cDNA. Ligation of the cDNA intopMHas5 was performed according to WO 01/12794. Briefly, 2 μl of vectorcorresponding to 40 ngs pMHas5 EcoRI-NotI restricted plasmid was usedwith 6 μl corresponding to about 100 ng of non size-fractionateddouble-stranded cDNA. pMHas5 2 μl cDNA 6 μl 10× lig. Buffer 1 μl T4 DNAligase (3U) 1 μl 10 μl

The ligation was incubated at 16° C. overnight and then heat-treated at65° C. for 20 minutes. 10 μl of dH2O was added to the ligation which wasthen stored at −20° C. until further use.

Rolling Circle Amplification

1 μl of ligated cDNA library corresponding to about 10 ng total DNA wasadded to 20 μl TempliPhi™ denaturing buffer (Amersham Biotech). Thesample was then heated to 95° C. for 3 minutes and then placed on ice.10 μl of TempliPhi™ premix was added to the sample which was incubatedfor 8 hours at 30° C. The sample was then heated to 95° C. for 5minutes, cooled at 4° C. and stored at −20° C. until further use.

3 μl sample material was restricted with NotI in a total volume of 10 μlunder standard conditions (New England Biolabs). 1 μl of the restrictiondigest mix was visualized on a 1% TBE agarose gel. The gel indicatedthat amplification had occurred. As expected, a diffuse band between 2.8and 3.5 kb was observed with tailing into higher molecular weights. Thisindicated that plasmids with different size inserts were successfullyamplified by the method, and that cutting with the unique rarely cuttingenzyme NotI created linear monomer plasmids containing the inserts.

The remaining 9 μl of the restriction digest mix was purified on a GFX™column to remove buffer and enzyme. The sample was eluted from thecolumn with 50 μl buffer (10 mM Tris, pH8.0). 5 μl of the purifiedsample was analyzed by agarose gel electrophoresis to verify that theamplification product was still present. 20 μl of the purified samplewas then used in a standard ligation: GFX purified digest of material:20 μl 10× lig. Buffer 10 μl H2O 69 μl T4DNA ligase (3U/ul) 1 μl 100 μl

The ligated plasmid is expected to contain mainly open circular plasmidmonomers with insert. 930 pg of the ligation was used to transform E.coli DH10B electrocompetent cells. 10 pg diluted supercoiled pUCC19monomer DNA was used as a control. The following results were obtained:transformation transformants/ug DNA ligation 5.6 × 10⁸ pUC19 8.6 × 10⁸

This result, clearly demonstrates that a library can be made by in vitrorolling circle amplification. The lower transformation frequency of thetreatment results primarily from the fact that the ligation is relaxedplasmid DNA, and not supercoiled as is the pUC19 control. In theliterature, the drop in transformation efficiency is reported to bebetween 100- and 1000-fold, in keeping with what we observed.

Transposon Assisted Signal Trapping and Supercoiling

Transposition with a SigA2 transposon was carried out as described indetail in WO 01/77315, with the following modifications: A period of 2hours at 30° C. was used for the transposition reaction. Then the samplewas split into two portions. The first was treated with a DNAtopoisomerase (DNA gyrase) according to the manufacturers instructions(John Innes Enterprizes, England). The second half was left untreated.

It was expected that by supercoiling the plasmid through treatment witha DNA gyrase, one can increase the efficiency of transformation into E.coli by 100-fold. This expectation was confirmed.

Using RecA Recombinase to Create Monomers

An efficient way to create monomers from a rolling circle amplifiedlibrary is to treat the concatameric DNA with a recombinase enzyme, suchas the commercially available RecA. enzyme from E. coli. The result ofsuch a treatment is the looping out of plasmid monomers from the verylong linear, amplification products of rolling circle. The method hasthe advantage that it is not dependent on the use of restriction enzymesthat may inadvertently cut the inserted library DNA. The recombinasestep may be performed at either simultaneously with the rolling circleamplification, after the amplification, or after transposon treatment.

Reduction of Amplification Bias by Use of Vector Specific PrimerCombinations

Although this has not been observed to any large degree, in ourexperience, selective amplification bias may occur based on differencesin the availability of priming sites for the random oligomers used inthe Amersham TempliPhi™ premix. In order to reduce this potential bias,several primers specific to the cloning vector may be used. As describedin Dean et al. (2001, Rapid Amplification of plasmid and phage DNA usingPhi29 DNA, polymerase and multiply-primed rolling circle amplification.Genome Research 11:1095-1099), many primers, priming on both DNA strandsare preferable to a one or few primers. In a preferred embodiment, amixture of 10-20 or more primers are used with an even distribution onthe each strand of the cloning vector.

1-44. (canceled)
 45. A method for isolating a polynucleotide thatencodes a polypeptide of interest which comprises a signal sequence forsecretion or partial secretion, the method comprising the sequentialsteps of: a) providing a DNA or cDNA library from an organism producingthe polypeptide of interest, wherein the library is comprised in acircular vector and is produced in vitro without ultraviolet irradiationof the component polynucleotides; b) amplifying the library by rollingcircle amplification, thereby forming concatamers; c) inserting into thelibrary a DNA fragment comprising a promoterless and secretionsignal-less polynucleotide encoding a secretion reporter; d) introducingthe amplified library comprising the inserted DNA fragment into a hostcell; e) screening for and selecting a host cell that secretes orpartially secretes the active secretion reporter; and f) identifyingfrom the selected host cell the polynucleotide into which the secretionreporter was inserted, and isolating the polynucleotide; wherein stepsb) and c) may be performed in any order.
 46. The method of claim 45,wherein the DNA or the cDNA library is normalized.
 47. The method ofclaim 45, wherein the DNA library or cDNA library is derived from amicroorganism.
 48. The method of claim 47, wherein the microorganism isa fungus, a filamentous fungus or a yeast.
 49. The method of claim 47,wherein the microorganism is a bacterium.
 50. The method of claim 47,wherein the microorganism is an archaeon.
 51. The method of claim 45,wherein the DNA library or cDNA library is derived from a multicellularorganism.
 52. The method of claim 45, wherein the vector comprises atleast one restriction enzyme cleavage site and/or at least one cos siteand/or at least one recombination recognition site.
 53. The method ofclaim 45, wherein step c) is performed in vitro.
 54. The method of claim45, wherein the DNA fragment comprises a transposon.
 55. The method ofclaim 45, wherein the DNA fragment comprises an origin of replicationwhich is functional in the host cell.
 56. The method of claim 45,wherein the secretion reporter is a protein which, when secreted fromthe host cell, allows said cell to grow in the presence of a substancewhich otherwise inhibits growth of said cell.
 57. The method of claim56, wherein the secretion reporter is a □-lactamase or an invertase. 58.The method of claim 45, wherein the polynucleotide of the DNA-fragmentof steb (b) encodes a secretion reporter carrying an N-terminal peptidelinker which comprises a specific target site for proteolytic cleavage.59. The method of claim 45, wherein the amplified library concatamersare converted to monomers before performing step d).
 60. The method ofclaim 45 wherein the vector comprises at least one restriction enzymerecognition site, and the concatamers are converted to monomers byrestriction enzyme digestion and then circularized by ligation.
 61. Apolynucleotide encoding a polypeptide of interest, wherein saidpolynucleotide is isolated by the method of the present invention.
 62. Apolypeptide of interest which is encoded by the polynucleotide of claim61.
 63. A host cell comprising at least one copy of the polynucleotideof claim
 61. 64. A process for producing a polypeptide of interest,comprising cultivating the host cell of claim 64 under conditionssuitable for expressing the polynucleotide, wherein said host cellsecretes the polypeptide encoded by said polynucleotide into the growthmedium.